Mechanism Of Regulation Of Nitrogen Fixation By NifLA In Pseudomonas Stutzeri A1501

Pseudomonas stutzeri A1501 (formerly Alcaligenes faecalis A1501), is an associative nitrogen-fixing bacteria, which colonizes the rhizosphere of rice. This strain can fix nitrogen under microaerobic conditions in the free-living state. In this study, we present data on the mechanism of regulation of nitrogen fixation by NifLA in pseudomonas stutzeri A1501.A cosmid library of A1501 was constructed using SuperCosl Cosmid Vector kit, and two transformants containing nifLA-like were screened by colony PCR and Southern hybridization. The 7.7kb DNA fragment containing nifLA-like was subcloned( EMBL/GenBank: AJ297529). The sequence analysis revealed that the highest identity of 72% was observed with NifL of A.vinelandii and 82% with NifA of A.vinelandii. nifL gene is single copy in P.stutzeri A1501.P. stutzeri A1501 nifHp-lacZ fusion vector was transfered into the wild type strain A1501 by tri-parental mating assay, then 6-galactosidase activity was assayed after induced at different ammonium and oxygen concentration. The result suggested that the expression of nifH was repressed by ammonium and oxygen. In order to study the regulation function of nifLA of Pseudomonas stutzeri A1501, nifL or nifA mutants and recombinant plasmids of nifA or nifL (pVA3 and pVL) were constructed, respectively. The nifA mutant and nifL polar-mutant were Nif negative, whereas non-polar nifL mutant displayed some residual nitrogenase activity even under high ammonium and oxygen concentration. By assaying acetylene reduction activity of transconjugants, the results showed that plasmid which expressed nifA from the Km promoter restored 30% nitrogenase activity to the nifA mutant strain, and this plasmid also increased nitrogenase activity of the wild type A1501 and the nifL non-polar mutant. The plasmid which constitutively expressed nifL descreased nitrogenase activity of A1501 and the nifL non-polar mutant. Also overexpressed NifA increased expression of nifHp-lacZ and overexpressed NifL couldn't work on nif gene expression.In order to study the interaction between NifL and NifA, full-length nifA and nifL and different regions encoding the open reading frame of nifLA in P. stutzeri A1501 were amplified by PCR with specific primers and cloned into plasmid pGAD-C(1)and pGBD-C(1). Recombinant plasmids were co-transformed into the expression host S. cerevisiae PJ69-4A by the lithium acetate method, and selected transformants were grown on SD plates lacking His/Ade, Leu, and Trp. An interaction between NifL and NifA in vivo was detected by yeast two-hybrid system, and it was shown that only the C-terminal domain of NifL displayed binding activity to NifA.Contraction of pET28a-nifA and pGEX2T-nifLc fusion expression vector were performed and transformed it into E. coli BL21(DE3) cells. Overexpression in soluble form was achieved with isopropyl β-D-thiogalactoside(IPTG). Interaction between NifA and NifL in vitro was proved using purified NifA and NifLc by binding assay. The result suggested that there existed a regulation mode that nif genes were inactivated by interaction between NifL and NifA.The nifLAp-lacZ fusion vector was constructed and transfered into the wild type strain A1501 and different mutants. The result of β-galactosidase assays suggested the nifLA promoter was dependent onNtrC and σ54, not absolutely on NifA and NifL.